This invention relates to the field of gel electrophoresis, and specifically to an apparatus and method for the elution and recovery of molecules isolated in such gels.
Gel electrophoresis is a technique for separating charged molecules having different charges or different molecular weights. It is commonly used for biological molecules, including proteins and nucleic acid polymers (such as DNA and RNA).
In gel electrophoresis, the commonly used gels are agarose gels and polyacrylamide gels. The gel may be thought of as a complex network of polymer molecules in which the channels between the molecules are occupied by a liquid such as a buffered aqueous solution. Electrodes are placed in a buffered aqueous solution, and the gel (which is also in the solution) is interposed between the electrodes. In effect, the gel is interposed in the current flow path between the electrodes and it separates the aqueous buffer solution in which the anode is placed from the aqueous buffer solution in which the cathode is placed.
A well or indentation is ordinarily provided in the gel, into which the sample is introduced. When a potential is applied to the electrodes, an electrical field is created through the gel and, under the influence of that field, charged molecules in the sample move through the gel. For example, negatively charged DNA molecules move through the gel toward the cathode. The rate of movement through the gel depends upon the molecular weight of the molecule. Migrating macromolecules must pass through a labyrinth of passageways in the gel. Because small molecules can traverse such a maze more rapidly than larger molecules, the rate of migration through the gel depends on the molecular weight of the molecule. For unknown reasons, the distance "D" moved by a molecule having molecular weight "M" depends logarithmically on M, according to the equation: EQU D=a-b logM,
in which a and b are empirically determined constants which depend on the temperature, the buffer used, and the makeup of the gel. As a result of such differential migration of molecules through the gel, sample molecules of different molecular weights become distributed throughout the gel in "bands". The electrophoresis process is ordinarily discontinued while the molecules of interest are located in a band or bands in the gel and before those molecules leave the gel and enter the buffer solution on the other side of the gel.
At that point in the process, the bands may be visualized by any appropriate visualization technique, such as by staining or by radioactive labeling and visualization. The gel is then physically cut into sections which contain only one band, and the sections containing molecules of interest are retained.
In order to recover the molecules at this point, it is necessary to elute them from the gel.
Prior art electroelution devices typically employ two large baths of buffer, each bath containing an electrode. The gel slice from which the molecule of interest is to be eluted ordinarily has been interposed between the electrodes in such a way that is creates a fluid seal between the baths. When a potential is applied to the electrodes, the molecule moves through the gel toward the electrode having a charge opposite to the charge of the molecule. This movement takes it out of the gel and into the buffer solution. The volume of the two buffer baths, in comparison to the volume of the gel, is high. The ratio of bath volume to gel volume is usually over 1000:1. Because of the large volume of buffer in the bath into which the molecule is eluted, various methods have been devised in the prior art to trap or catch the molecule of interest. For example, in U.S. Pat. No. 3,969,218, molecules leaving the gel are shunted by flowing liquid away from the electrode and into a separate receptacle. U.S. Pat. No. 4,049,534 discloses trapping the eluted molecules with a dialysis membrane interposed between the gel and the electrode. In U.S. Pat. No. 4,545,888, filter discs of DEAE cellulose are provided between the gel and the electrode to trap eluted DNA molecules. One commercial device, the International Biotechnologies, Inc. electroelutor, seeks to trap the eluted molecules in a saline solution interposed between the gel and the electrode.
The elution time in these devices is often between twenty minutes and one hour. During elution, substantial electrolysis with attendant bubble generation occurs. Moreover, the buffer solutions and the gels are heated by the current flow between the electrodes. This has the potential for damaging the biological molecules being eluted. For this reason, the baths in which the electrodes are placed are ordinarily very large, in order to provide a heat sink capability.
Great pains have been taken in the prior art to prevent contact between the eluted molecules and the electrodes. It is apparently the common understanding in the art that such molecule-electrode contact can lead to degradation or other damage to the molecule.